Metastatic breast cancer is the leading cause of death among women between the ages of 15 and 54 and affects approximately 13% of women during their lifespan. These can be grossly categorized as ductal or lobular depending on their site of origin in normal breast tissue. Tumors usually begin as non-invasive cells at the site of tumor origin, spread to surrounding tissue in the breast and eventually become fully metastatic and migrate to the lymph nodes and other parts of the body.
There is increasing evidence that cell-cell adhesion is a potent suppressor of metastatic breast cancer progression (Berx and Van Roy, 2001). For example, in infiltrating lobular breast carcinomas E-cadherin is often lost and the resulting disruption of adherens junctions initiates a complete dissolution of cell-cell adhesion which allows single cells to break away from the primary tumor and invade the stroma in a single file pattern (Cleton Jansen et al., 2002). Alterations in cell adhesion are more subtle in infiltrating ductal carcinomas where invasion is characterized by the movement of clusters of cells into the stroma (Page and Simpson, 2000). In the latter situation adherens junctions are often present (Acs et al., 2001; Gillett et al., 2001) but there appears to be a general loss of polarity that is characterized by the mislocalization of apical markers such as MUC-1 (McGuckin et al., 1995; Mommers et al., 1999; Diaz et al., 2001; Rahn et al., 2001) that may be fueled by the disruption of tight junctions (Hoover et al., 1997; Kramer et al., 2000; Kominsky et al., 2003). While transcriptional repressors of E-cadherin expression have been identified (Cano et al., 2000; Guaita et al., 2002), little is known about the mechanism responsible for the disruption of tight junctions during breast tumor progression.
In adult vertebrates, blood homeostasis is maintained by a pool of predominantly quiescent (Cheshier et al., 1999), multipotent hematopoietic stem cells (HSC) and more mature hematopoietic progenitors cells (HPC) that reside in specific microenvironmental niches in the bone marrow (reviewed by Scadden (Scadden, 2006)). These hematopoietic cells have an intrinsic homing mechanism to facilitate their retention and maintenance in this microenvironment (Whetton and Graham, 1999). Furthermore, when appropriately triggered to mobilize to the circulation (perhaps by host injury), HSC/P homing allows these cells to re-localize to the appropriate niche once blood homeostasis has been restored (Nervi et al., 2006). In the clinic, this homing property is exploited in everexpanding hematopoietic stem cell transplant (HSCT) (Thomas et al., 1957) treatment modalities for leukemia and lymphoma (Mehta and Davies, 2008; Shenoy and Smith, 2008; Sierra et al., 2008; Stein and Forman, 2008; Tse et al., 2008), autoimmune disorders (Alderuccio et al., 2006), inherited immunodeficiency and metabolic disorders (Dvorak and Cowan, 2008; Prasad and Kurtzberg, 2008), hemaglobinopathies (Pinto and Roberts, 2008; Ullah et al., 2008) and various forms of bone marrow failure (Barrett and Savani, 2008; Gluckman and Wagner, 2008; Perez-Albuerne et al., 2008). Successful treatment relies on the long-term engraftment of the donor HSCs, which, in turn, demands a faithful execution of a complex progression of cell movements including: vascular adhesion, diapedesis, migration towards a chemokine gradient and then adhesion and stable lodgment of donor HSC to the niche (Hart et al., 2004; Kaplan et al., 2007).
The processes of homing and engraftment are directed and influenced by a variety of constituents (reviewed by Laird, von Andrian and Wagers (Laird et al., 2008) and Kaplan, Psaila and Lyden (Kaplan et al., 2007)) some of these include: cell-cell and cell-matrix adhesion molecules and their ligands, soluble and cell-bound chemoattractants and survival factors, proteases, extracellular matrix components and extracellular calcium ion (Ca2+) (Adams et al., 2006). When in the bone marrow circulation, HSCs initiate tethering and rolling via interaction with selectins and subsequently sense a chemotactic gradient that, when combined with additional cell-adhesion signals, lead to diapedesis through the bone marrow vasculature followed by directional chemotaxis to reach the niche (Papayannopoulou and Craddock, 1997; Williams et al., 1991). This α4-integrin mediated migration is well coordinated in a way that permits cells to adhere and yet also to remain mobile and migratory. Accordingly, equilibrium is established between activated and non-activated integrins at the cell surface in order to propel the cell forward during this migration.
CXCL12 (formerly known as stromal-derived factor or SDF-1α) is a vital chemoattractant in HSC/P homing that is produced by cells of the bone marrow stroma—a term describing a heterogeneous component of the bone marrow niche (Cheshier et al., 1999). Alone or in concert with other factors, CXCL12 has pleiotropic effects on HSC/Ps including modulation of motility, regulation of HSC homing to and retention within the bone marrow niche, promotion of survival (Broxmeyer, 2008; Lee et al., 2002); and, stimulation of proliferation (Aiuti et al., 1997; Aiuti et al., 1999). CXCL12 exerts is effects via CXCR4, a seven-pass transmembrane G protein-coupled receptor (GPCR) that is expressed by diverse immature and mature blood cells (Broxmeyer, 2008; Tavor et al., 2004; Weissman, 1996). In addition to playing a role in the normal trafficking of a variety of blood cell types, CXCR4 has also been shown to play an important role in tumor cell metastasis (Burger and Kipps, 2006; Burger and Burkle, 2007; Hartmann et al., 2005). There is considerable evidence that the CXCR4-CXCL12 axis is central to maintaining the HSC pool in the marrow niche. For example, inducible deletion of CXCR4 in adult mice causes HSC depletion and increased sensitivity to 5-fluorouracil (Sugiyama et al., 2006) and small molecule CXCR4-specific agonists, CXCL12-mimetic peptides (recently reviewed by Pelus and Fukuda (Pelus and Fukuda, 2008)), antibodies to CXCR4, and, inhibitors of CXCL12 proteolytic activation (Campbell and Broxmeyer, 2008) can all induce mobilization of hematopoietic precursor cells to the peripheral blood or block homing to the bone marrow.
Another factor produced by stromal cells of the bone marrow niche is stem-cell factor (SCF), an essential factor in hematopoiesis that binds and activates the receptor tyrosine kinase c-Kit (Blechman et al., 1993; Blechman and Yarden, 1995; Broudy, 1997). Expressed as either a secreted soluble factor or a membrane-bound factor, SCF not only aids in the homing and maintenance of HSCs (Driessen et al., 2003) within the niche, but also the survival and proliferation of HSCs (Hart et al., 2004). Since SCF and CXCL12 exert both distinct and overlapping effects on hematopoietic cells and share many of the same intracellular signaling pathways to mediate their effects, they work together to enhance HSC cell motility, proliferation and survival (Cancelas et al., 2006; Glodek et al., 2007; Kapur et al., 2001; Williams et al., 2008).
CD34 was initially identified over 20 years ago as an hematopoietic stem cell and vascular endothelial marker and has alternatively been proposed to act as an: 1) enhancer of proliferation, 2) a blocker of differentiation, 3) bone marrow homing receptor, 4) cell adhesion molecule, and 5) a blocker of cell adhesion (Fackler et al, 1996, Krause et al. Blood, 1996, Baumhueter et al. 1993). The CD34 antigen has long been used as a marker to identify and enrich donor-derived HSC with long-term repopulating potential in clinical applications of HSCT. CD34 is the founding member of a family of related HSC sialomucins including podocalyxin and endoglycan (Furness and McNagny, 2006) (Nielsen JS and McNagny J Cell Sci. 2008 Nov. 15; 121(Pt 22):3683-92). In mice, CD34 is expressed by a subset of mature blood cells and immature progenitors as well as all vascular endothelia including specialized endothelial cells (termed high endothelial venules or HEV) in lymph nodes. CD34 gene knockout mice are relatively normal with very subtle defects in hematopoietic and vascular function. The function of CD34 has been widely debated, but the current data suggest that it (and its relatives) can either promote (Baumheter et al., 1993; Hiraoka et al., 1999; Puri et al., 1995; Sassetti et al., 1998b) or obstruct cell-cell adhesion interactions (Blanchet et al., 2007; Drew et al., 2002; Drew et al., 2005) depending on the context and tissue type (reviewed in Nielsen and McNagny, J Cell Sci. 2008 Nov. 15; 121(Pt 22):3683-92). The most clear-cut experiments suggest that CD34-type proteins can act as either pro-adhesive or anti-adhesive molecules depending on their glycosylation status (Satomaa, 2002, Baumhueter et al., 1993 and Bistrup et al., 1999).
Additional evidence for an anti-adhesive function for this family of molecules comes from mutational analysis of CD34's closest relative, podocalyxin. Podocalyxin, which was named for its prominent expression on kidney podocytes (Dekan et al., 1991; Horvat et al., 1986; Kerjaschki et al., 1984) is also expressed by HSCs and all vasculature.
Podocalyxin, (also called podocalyxin-like protein 1 (PCLP-1), Myb-Ets-transformed progenitor (MEP21) or thrombomucin) is a heavily sialylated and sulfated integral membrane glycoprotein that interacts with the actin cytoskeleton. It belongs to the CD34 family of sialomucins and is highly expressed on the surface of hematopoeitic progenitors, vascular endothelia and podocytes which form a tight junction-free epithelial meshwork that surrounds glomerular capillaries in the kidney (Kerjaschki et al., 1984; Kershaw et al., 1995; McNagny et al., 1997). Evidence suggests that the primary function of this molecule is to act as a type of molecular “Teflon™” to block inappropriate cell adhesion. For example, as kidney podocytes begin to express podocalyxin they undergo a dramatic morphological shift from adherent, tight junction-associated monolayers surrounding the glomerular capillaries to a more modified cell layer lacking tight junctions and with extensive fully-interdigitated foot processes that are separated from each other by slit diaphragms. These podocalyxin-covered slit diaphragms form the primary filtration apparatus of the kidney. Deletion of the podocalyxin-encoding gene in mice results in the persistence of tight-junctions between podocytes, a lack of foot process formation and perinatal death due to anuria and high blood pressure (Doyonnas et al., 2001). In this context, podocalyxin acts as an anti-adhesive to facilitate the dissolution of cell-cell junctions and drive the formation of the extensive podocyte foot processes required for renal filtration (Doyonnas et al., 2001). Conversely, when podocalyxin is ectopically expressed in kidney epithelial cell monolayers, tight junctions and adherens junctions are both disrupted (Takeda et al., 2000). In this context, podocalyxin decreases cell-cell adhesion by expanding the apical cell domain and marginalizing junctional complexes between cells in monolayers (Takeda et al., 2000). Thus, both gain-of-function and loss-of-function experiments suggest that podocalyxin acts as a tissue-specific anti-adhesin during normal kidney development (Takeda et al., 2001, Doyonnas et al., 2001).
Circumstantial evidence suggests that podocalyxin expression may be upregulated in a variety of neoplastic scenarios. For example podocalyxin was recently identified as the peanut agglutinin-binding tumor antigen gp200 expressed on human embryonal carcinomas. (Schopperle et al., 2002). In addition, the human podocalyxin gene (PODXL) has been assigned to chromosome 7q32-q33 (Kershaw et al., 1997), which places PODXL very close to the 7q35ter region that has been identified as a gain site by comparative genomic hybridization in ductal carcinoma in situ, infiltrating ductal carcinoma and in lymph node metastasis (Aubele et al., 2000). Thus, while it is not yet clear whether the PODXL gene is amplified in breast carcinoma, its expression may be unduly influenced by a nearby amplicon. Under anemic conditions the inventors have recently shown that Podocalyxin expression is upregulated in mouse erythroid progenitor cells (McNagny submitted unpublished obs). Therefore, podocalyxin expression may be similarly upregulated in necrotic breast carcinomas where hypoxia-regulated genes are transcriptionally activated (Adeyinka et al., 2002). If this is indeed the case, it would have functionally important implications as tumor hypoxia helps to drive solid tumor progression generally (Knowles and Harris, 2001) and ductal carcinoma progression specifically (Bos et al., 2003; Helczynska et al., 2003). Up-regulated podocalyxin expression has been found to mark the most invasive human epithelial tumours of prostate and breast (Casey et al., 2006; Sizemore et al., 2007; Somasiri et al., 2004).
Podocalyxin expression has been detected on a limited set of non-cancerous hematopoietic cells in adult mammals including activated platelets (Miettinen et al., 1999), anemia-induced stress reticulocytes and erythroblasts (Doyonnas et al., 2005; Sathyanarayana et al., 2007), and, importantly, on a subset of primitive bone marrow resident hematopoietic progenitors with long-term repopulating capacity (Doyonnas et al., 2005). Despite its limited expression in mammalian adult hematopoietic tissue, podocalyxin is highly-expressed on the surface of definitive hematopoietic cells derived from the fetal liver of E15.5 mouse embryos (Doyonnas et al., 2005). This expression is first detected in hematopoietic progenitors and primitive erythroid progenitors of the yolk sac and is maintained on multi-potential blood progenitors and multi-lineage hematopoietic cells of embryonic fetal liver. Podocalyxin expression declines dramatically before birth and then is expressed again at high levels for a brief window as adult hematopoiesis is established in the marrow (Doyonnas et al., 2005).
The present inventors have previously hypothesized that podocalyxin behaves as a regulator of cell adhesion during hematopoietic cell migration or hematopoietic stem/progenitor cell engraftment since its expression correlates with the ontogenetical migration and engraftment of HSCs in developing mouse embryos (Doyonnas et al., 2005), and, because it is expressed on a subset of lineage-sca-1+c-kit+ (LSK) cells with enhanced long-term HSC-repopulating potential (Doyonnas et al., 2005).
Using homologies present in the cytoplasmic tails of CD34 and podocalyxin, endoglycan was identified as a novel member of this family of glycoproteins. Endoglycan mRNA and protein were detected in both endothelial cells and CD34+ bone marrow cells (Sassetti et al., 2000). Endoglycan, like CD34 and podocalyxin can function as a L-selectin ligand. Endoglycan utilizes a different binding mechanism, interacting with L-selectin through sulfation on two tyrosine residues and O-linked sLex structures (Fieger et al., 2003).